CN106011132A - Method for extracting corn or radish genome DNA with high flux - Google Patents

Method for extracting corn or radish genome DNA with high flux Download PDF

Info

Publication number
CN106011132A
CN106011132A CN201610672468.4A CN201610672468A CN106011132A CN 106011132 A CN106011132 A CN 106011132A CN 201610672468 A CN201610672468 A CN 201610672468A CN 106011132 A CN106011132 A CN 106011132A
Authority
CN
China
Prior art keywords
buffer
semen maydis
dna
orifice plate
high flux
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN201610672468.4A
Other languages
Chinese (zh)
Inventor
陈发波
袁亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yangtze Normal University
Chongqing Academy of Agricultural Sciences
Original Assignee
Yangtze Normal University
Chongqing Academy of Agricultural Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yangtze Normal University, Chongqing Academy of Agricultural Sciences filed Critical Yangtze Normal University
Priority to CN201610672468.4A priority Critical patent/CN106011132A/en
Publication of CN106011132A publication Critical patent/CN106011132A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method for extracting corn or radish genome DNA with high flux. The method is characterized in that a leaves sample of corn or radish to-be-detected can be obtained with batches, then alkali lye is added, cell wall and cell membrane of the plant leaves can be destroyed under high temperature condition, DNA is released, an acidic neutralization extract is added so that the sample is suitable for PCR amplification, and then an amplification reaction is carried out to realize detection of target molecule. The method has the advantages that operation is simple, the method is benefit for manual operation, is suitable for operation by a miniature laboratory, and the processing efficiency is high.

Description

A kind of high flux extracts Semen Maydis or the method for radish gene group DNA
Technical field
Agriculture plant breeding DNA extraction technical field of the present invention, is specifically related to a kind of high flux and extracts Semen Maydis or radish gene group The method of DNA.
Background technology
Crop breeding, also known as breed improvement, is by creatingHereditary variation, improvement inherited character, to cultivate excellent animals and plants New varieties so that it is reach the technology of high yield, stable yields, high-quality, excellent results.To developing animal husbandry and plant husbandry has particularly significant Meaning.During crop breeding, use is generally individually needed to arrivePolymerase chain reactionTechnology realizes molecular marker,Polymerase chain Formula is reacted(PCR) it is a kind of for amplifying the specific DNA fragmentation of amplificationProtocols in Molecular Biology, it is considered as in vitro Special DNA replication dna, the maximum feature of PCR, is to be significantly increased by the DNA of trace.
Semen Maydis based on polymerase chain reaction, Radix Raphani molecular mark need to identify in big colony and contain There are individual plant or the strain of objective trait (degeneration-resistant, high-quality).In molecular breeding is put into practice, one of method is that research worker is by breeding group Body is pressed simple grain and is germinateed in seedbed, and seedling completes the Molecular Identification to objective trait before moving into field planting.The two of method are Directly seed is carried out Molecular Detection.While it is true, research worker would generally be at a breeding generation to multiple breeding populations Thousands of parts of individual plants carry out the Molecular Identification of objective trait.Therefore, quick obtaining number becomes with thousand parts of DNA that can be used for PCR amplification One of factor of restriction molecule labelling efficiency of selection.
At present, the method extracting DNA of plants conventional has CTAB(cetyl trimethylammonium bromide) method, SDS(dodecyl Sodium sulfate) method such as method, DNA commercial reagents box.
CTAB method and SDS method major defect are that process is complicated, loaded down with trivial details time-consumingly.Extract Radix Raphani and maize dna needs 8 to 10 Step, every part of sample needs to be fully ground in liquefied ammonia, has cross-contamination unavoidably, need subsequently during grinding between sample Long-time concussion, is centrifuged repeatedly, extracts and washs, and is finally dried and dissolves.1 day multipotency of 1 skilled technician extracts 30 parts Sample, this is far from the needs meeting molecular mark practice.
DNA commercial reagents box major defect is expensive, and use cost is high.Molecule assisted Selection is applied to the master of breeding Want advantage can improve breeding efficiency, reduce breeding cost.But, utilize commercial reagents box to extract DNA and carry out Molecular Detection by bright The aobvious cost improving molecule assisted Selection, thus add breeding cost.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the technical problem to be solved is: how to provide a kind of for jade Rice, Radix Raphani DNA, simple to operate, beneficially manual operation, with low cost, and the high high flux for the treatment of effeciency extracts Semen Maydis or Radix Raphani base Because of the method organizing DNA.
In order to solve above-mentioned technical problem, present invention employs following technical scheme:
A kind of high flux extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that first obtain jade to be detected in bulk Rice or the leaf sample of Radix Raphani, be then respectively adding alkali liquor, destroy plant leaf blade cell wall and cell membrane under the high temperature conditions, Being discharged by DNA, the neutralising extract being subsequently adding acidity is allowed to be suitable for PCR amplification, then realizes mesh after carrying out amplified reaction Mark Molecular Detection.
This method, specifically includes following steps:
A, the seed simple grain of batch Semen Maydis to be detected or Radix Raphani is cultivated grow blade;
B, it is sampled according to the 30-60 square millimeter each Semen Maydis to cultivating or radish plant blade, and puts into process orifice plate In each aperture;
C, preparation buffer A and buffer B, buffer A is 0.1mol/L NaOH and 2% Tween20 equal-volume is mixed to get, Buffer B: 0.1mol/L Tris-HCl and 0.002 mol/L EDTA equal-volume are mixed to get;To each aperture of process orifice plate After middle addition 500ul buffer A, process orifice plate is heated a period of time;
D, again addition 500ul buffer B in each aperture of process orifice plate, shake a period of time so that liquid is sufficiently mixed, and releases Release DNA;
E, PCR reaction system is distributed in each aperture of reaction orifice plate, process orifice plate after concussion in corresponding removing step d During liquid enters into each aperture of reaction orifice plate in each aperture, carry out amplified reaction, then realize objective trait Molecular Detection.
The cardinal principle of this method is that alkalescence, the condition of high temperature can destroy plant leaf blade cell wall and cell membrane, thus will DNA discharges, and then is allowed to be suitable for PCR amplification with acid neutralising extract.Concentration Tween20 within 2% can crack carefully After birth released dna, again will not suppression PCR amplified reaction.Therefore this method need not be ground sample and pretreatment, for little The current demand of type laboratory, by the nucleic acid extraction of high flux low cost, under conditions of being not required to mechanical hand, can realize full The Molecular Detection of the high flux objective trait of foot breeding demand.
As optimization, in a step, the seed simple grain of batch Semen Maydis to be detected or Radix Raphani is respectively put into breeding hole In each aperture of plate, cultivate in illumination box and grow blade;Breeding orifice plate preferably employs 96 orifice plates.So can ensure that and criticize The efficiency that amount processes.
In b step, it is preferred to use plant leaf blade sampler is sampled, and process orifice plate preferably employs 96 hole PCR plate Guaranteed efficiency.
In step c, buffer is allocated and can be used following methods:
(1) 100mM NaOH: being dissolved in by 0.2 g NaOH in double pure water, constant volume is 50 ml;
(2) 2% Tween20: take 5 g 20% Tween20 and be dissolved in double pure water of 50 ml;
(3) 100mM Tris-HCl: taking 0.605 g Tris-HCl and be dissolved in double pure water, regulate PH=8, constant volume is 50ml;
(4) 2mM EDTA: taking 0.037 g EDTA and be dissolved in double pure water, regulate PH=8, constant volume is 50ml;
(5) buffer A: 0.1mol/L NaOH and the mixing of 2% Tween20 equal-volume, now with the current;
(6) buffer B: 0.1mol/L Tris-HCl and the mixing of 0.002 mol/L EDTA equal-volume.
Use said method, convenient enforcement to allocate, be especially advantageous in small test room environmental implementing preparation.
In step c, it is preferred to use the 12 passage liquid-transfering guns of range 1000ul add buffer A, to facilitate operation and to improve Efficiency;Wherein heating purpose is to utilize alkalescence, the condition destruction plant leaf blade cell wall of high temperature and cell membrane, thus is released by DNA Release, it is preferred to use in thermal cycler, 95 DEG C of heating 10min, can just reach this effect and be unlikely to again to destroy DNA.
In Step d, use and on earthquake device, shake 5min, make buffer A and buffer B be sufficiently mixed;To ensure mixing Fully, DNA can fully be discharged in solution.
In step e, reaction orifice plate preferably employs 96 hole PCR plate guaranteed efficiencies;Liquid pipettes and preferably employs range The 12 passage liquid-transfering guns of 1000ul, to facilitate operation and to improve efficiency;0.1% can also be added further in PCR reaction system Bovine serum albumin (BSA) and 1% polyvinylpyrrolidone (PVP) can effectively reduce the effect of inhibitive factor, improve detection effect Really.
This method has the following characteristics that 1, most important advantage is high flux and low cost.Utilize the method extract DNA, 1 Individual research worker can process more than 2000 sample for 1 day, and the cost extracting 1 DNA is approximately 0.05 dollar.Than CTAB and SDS method Save time, more cheap than test kit.2, the blade amount needed is few.It is about 30-60mm2Enough 100 PCR of DNA that extract of blade expand Increase reaction.3, while having above-mentioned advantage, the DNA that the method is extracted can stably preserve 1 month, at-20 DEG C at 4 DEG C Stably preserve 3 months.4, extract DNA by the method, carry out pcr amplification reaction in this, as template, be not required to Robot actions In the case of, it is possible to realize sequencing and the standardization of Molecular Detection.Therefore, application the method extraction DNA is very suitable for little Type labs molecular marker assisted selection works.
In sum, the present invention has simple to operate, beneficially manual operation, and applicable small-size laboratory is carried out, with low cost, And treatment effeciency advantages of higher.
Accompanying drawing explanation
Fig. 1 is to take Radix Raphani DNA extraction sample liquid during Radix Raphani verification experimental verification to carry out agarose gel electrophoresis detection, different turnip leaves The result schematic diagram of the extracting solution detection of taken amount.
Fig. 2 is to take Radix Raphani DNA extraction sample liquid during Radix Raphani verification experimental verification to carry out agarose gel electrophoresis detection, and sampling amount is 5mg Radix Raphani DNA extraction liquid agarose gel electrophoresis figure.
Radix Raphani DNA extraction liquid PCR primer detection figure when Fig. 3 is Radix Raphani verification experimental verification.
Fig. 4 is to take maize dna extraction sample liquid during corn trials checking to carry out agarose gel electrophoresis detection, different leaf of Semen Maydis The result schematic diagram of the extracting solution detection of sheet taken amount.
Fig. 5 is to take maize dna extraction sample liquid during corn trials checking to carry out agarose gel electrophoresis detection, and sampling amount is The maize dna extracting solution agarose gel electrophoresis figure of 10mg.
Maize dna extracting solution sepharose electrophoresis detection figure when Fig. 6 is corn trials checking.
Detailed description of the invention
The present invention is described in further detail with optimum embodiment below in conjunction with the accompanying drawings.
Optimum embodiment: a kind of high flux extracts Semen Maydis or the method for radish gene group DNA, specifically includes following steps:
A, the seed simple grain of batch Semen Maydis to be detected or Radix Raphani is cultivated grow blade;
B, it is sampled according to the 30-60 square millimeter each Semen Maydis to cultivating or radish plant blade, and puts into process orifice plate In each aperture;
C, preparation buffer A and buffer B, buffer A is 0.1mol/L NaOH and 2% Tween20 equal-volume is mixed to get, Buffer B: 0.1mol/L Tris-HCl and 0.002 mol/L EDTA equal-volume are mixed to get;To each aperture of process orifice plate After middle addition 500ul buffer A, process orifice plate is heated a period of time;
D, again addition 500ul buffer B in each aperture of process orifice plate, shake a period of time so that liquid is sufficiently mixed, and releases Release DNA;
E, PCR reaction system is distributed in each aperture of reaction orifice plate, process orifice plate after concussion in corresponding removing step d During liquid enters into each aperture of reaction orifice plate in each aperture, carry out amplified reaction, then realize objective trait Molecular Detection.
In present embodiment, in a step, the seed simple grain of batch Semen Maydis to be detected or Radix Raphani is respectively put into breeding With in each aperture of orifice plate, cultivate in illumination box and grow blade;Breeding orifice plate uses 96 orifice plates.In b step, employing is planted Thing blade sampler is sampled, and process orifice plate uses 96 hole PCR plate.In step c, buffer allotment can use below Method:
(1) 100mM NaOH: being dissolved in by 0.2 g NaOH in double pure water, constant volume is 50 ml;
(2) 2% Tween20: take 5 g 20% Tween20 and be dissolved in double pure water of 50 ml;
(3) 100mM Tris-HCl: taking 0.605 g Tris-HCl and be dissolved in double pure water, regulate PH=8, constant volume is 50ml;
(4) 2mM EDTA: taking 0.037 g EDTA and be dissolved in double pure water, regulate PH=8, constant volume is 50ml;
(5) buffer A: 0.1mol/L NaOH and the mixing of 2% Tween20 equal-volume, now with the current;
(6) buffer B: 0.1mol/L Tris-HCl and the mixing of 0.002 mol/L EDTA equal-volume.
In present embodiment step c, the 12 passage liquid-transfering guns of range 1000ul are used to add buffer A.Use thermal cycle 95 DEG C of instrument heating 10min.In Step d, use and on earthquake device, shake 5min, make buffer A and buffer B be sufficiently mixed.e In step, reaction orifice plate uses 96 hole PCR plate, and liquid pipettes the 12 passage liquid-transfering guns preferably employing range 1000ul.Simultaneously 0.1% bovine serum albumin and 1% polyvinylpyrrolidone is added in PCR reaction system.
Below, it is respectively directed to radish seed and corn seed, based on above-mentioned detailed description of the invention operating procedure, uses Tests below verifies effect of the present invention.
One, Radix Raphani verification experimental verification.
The extraction of Radix Raphani DNA and detection.
1, radish seed is put in incubator germinate, blade sampling gradient be preset as 2 mg, 5 mg, 10 mg, 15 mg, 20 mg、25 mg、30 mg、35 mg、40 mg、50 mg。
2, DNA extraction is completed by the operating procedure in detailed description of the invention.
3, DNA detection: take Radix Raphani DNA extraction sample liquid and carry out agarose gel electrophoresis detection.It is illustrated in figure 1 different Folium Raphani The extracting solution detection figure of sheet taken amount, in figure: M represents DNAmarker;;1~10 loading wells are all the different sampling amounts of same volume DNA extraction liquid, be followed successively by from left to right 50 mg, 40 mg, 35 mg, 30 mg, 25 mg, 20 mg, 15 mg, 10 mg, 5 Mg, the DNA extraction liquid of turnip leaves taken amount of 2 mg.As shown in Figure 1: blade sampling amount does not the most affect the dense of DNA in extracting solution Degree.
Fig. 2 be sampling amount be the Radix Raphani DNA extraction liquid agarose gel electrophoresis figure of 5mg, in figure: M represents marker;1~13 Loading wells is followed successively by extracting solution 6.0 μ l, 5.75 μ l, 5.5 μ l, 5.25 μ l, 5.0 μ l, 4.75 μ l, 4.5 μ l, 4.25 μ l、4.0 μl、3.75 μl、3.50 μl、3.25 μl、3.0 μl.As seen from the figure: utilize this method can extract Radix Raphani DNA, extract In liquid, the concentration containing the DNA, DNA discharged increases along with the extracting liquid volume of detection and improves.
4, the pcr amplification reaction with this DNA as template.
PCR reaction system (25 μ l): 12.5 μ l 2 × Taq Master Mix, ITSR upper and lower primer (10 nmol/ Ml) each 1 μ l, DNA extraction liquid 3 μ l, Nase Free Water 7.5 μ l.
PCR response procedures:
5, amplified reaction electrophoresis detection is carried out, as it is shown on figure 3, be Radix Raphani DNA extraction liquid PCR primer detection figure, in figure: M represents marker;1-5 is followed successively by blade sampling amount 5 mg; 10 mg;15 mg ;20 mg; 25 mg ;The primer is ITSR, The band of its amplification is it is contemplated that 750bp.As shown in Figure 3: the DNA extraction liquid of all taken amounts can amplify expection product, PCR expands Agarose gel electrophoresis effect and the blade sampling amount of volume increase thing number unrelated.The DNA extraction liquid that the method is obtained as Pcr template, it is possible to amplify expection band, it was demonstrated that the method feasibility in Radix Raphani Molecular Detection.
Two, corn trials checking.
The extraction of maize dna and detection.
1, corn seed is put in incubator germinate, blade sampling gradient be preset as 2 mg, 5 mg, 10 mg, 15 mg, 20 mg、25 mg、30 mg、35 mg、40 mg、50 mg。
2, DNA extraction is completed by the operating procedure in detailed description of the invention.
3, DNA detection: take maize dna extraction sample liquid and carry out agarose gel electrophoresis detection.It is not that result sees Fig. 4, Fig. 4 DNA agarose gel electrophoresis figure is extracted with blade taken amount.In figure: M represents DNA marker;1~10 loading wells are all identical The DNA extraction liquid of volume difference sampling amount.Be followed successively by from left to right 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 Mg, 35 mg, 40 mg, the DNA extraction liquid of different maize leaf taken amounts of 50 mg.As shown in Figure 4: all blade taken amounts DNA carry Take liquid and all can detect DNA.And can be seen that the extracting solution DNA concentration of 10 mg blades is the highest.Fig. 5 be sampling amount be 10mg's Maize dna extracting solution agarose gel electrophoresis figure.In figure: M represents DNAmarker;1-6 is followed successively by extracting solution 5 μ l, 4.5 μ L, 4 μ l, 3.5 μ l, 3 μ l, 2.5 μ l, 7 bromophenol blues.As shown in Figure 5: pipette 4 μ l DNA extraction liquid and carry out Detection results Best.
4, the pcr amplification reaction with this DNA as template.
PCR reaction system (25 μ l): 12.5 μ l 2 × Taq Master Mix, ITSR upper and lower primer (10 nmol/ Ml) each 0.5 μ l, DNA are 1 μ l, Nase Free Water 10.5 μ l.
PCR response procedures:
5, amplified reaction electrophoresis detection.Fig. 6 is maize dna extracting solution sepharose electrophoresis detection figure;In figure: M represents DNAmarker;1~10 be followed successively by blade sampling amount 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg、50 mg.The primer is ITSR, and it amplifies band it is contemplated that 750bp.As shown in Figure 6: the DNA of all taken amounts carries Taking liquid and can amplify expection product, sample taken amount is 2 mg, 5 mg, 10 mg also can amplify product, but be not reaching to pre- Phase effect.Can be seen that the PCR amplification of Semen Maydis is relevant to blade sampling amount, and be gradually increased to a certain extent at blade sampling amount Time on PCR reaction impact little.Semen Maydis extracts the leaf quality of DNA preferably between 10 mg~30 mg.
Test Summary: to sum up, utilizes the method can extract DNA from Semen Maydis, Radix Raphani, and the DNA extracted disclosure satisfy that PCR The requirement of amplified reaction, small-size laboratory uses the method, it is possible to realize high flux and low cost.

Claims (10)

1. a high flux extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that first obtain to be detected in bulk Semen Maydis or the leaf sample of Radix Raphani, be then respectively adding alkali liquor, destroys plant leaf blade cell wall and cell under the high temperature conditions Film, discharges DNA, and the neutralising extract being subsequently adding acidity is allowed to be suitable for PCR amplification, then carries out realization after amplified reaction Target molecule detects.
2. high flux as claimed in claim 1 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that specifically include Following steps:
A, the seed simple grain of batch Semen Maydis to be detected or Radix Raphani is cultivated grow blade;
B, it is sampled according to the 30-60 square millimeter each Semen Maydis to cultivating or radish plant blade, and puts into process orifice plate In each aperture;
C, preparation buffer A and buffer B, buffer A is 0.1mol/L NaOH and 2% Tween20 equal-volume is mixed to get, Buffer B: 0.1mol/L Tris-HCl and 0.002 mol/L EDTA equal-volume are mixed to get;To each aperture of process orifice plate After middle addition 500ul buffer A, process orifice plate is heated a period of time;
D, again addition 500ul buffer B in each aperture of process orifice plate, shake a period of time so that liquid is sufficiently mixed, and releases Release DNA;
E, PCR reaction system is distributed in each aperture of reaction orifice plate, process orifice plate after concussion in corresponding removing step d During liquid enters into each aperture of reaction orifice plate in each aperture, carry out amplified reaction, then realize objective trait Molecular Detection.
3. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in a step, The seed simple grain of batch Semen Maydis to be detected or Radix Raphani is respectively put in each aperture of breeding orifice plate, in illumination box Cultivation grows blade;Breeding orifice plate uses 96 orifice plates.
4. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in b step, Use plant leaf blade sampler to be sampled, and process orifice plate uses 96 hole PCR plate.
5. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in step c, Buffer is allocated and can be used following methods:
(1) 100mM NaOH: being dissolved in by 0.2 g NaOH in double pure water, constant volume is 50 ml;
(2) 2% Tween20: take 5 g 20% Tween20 and be dissolved in double pure water of 50 ml;
(3) 100mM Tris-HCl: taking 0.605 g Tris-HCl and be dissolved in double pure water, regulate PH=8, constant volume is 50ml;
(4) 2mM EDTA: taking 0.037 g EDTA and be dissolved in double pure water, regulate PH=8, constant volume is 50ml;
(5) buffer A: 0.1mol/L NaOH and the mixing of 2% Tween20 equal-volume, now with the current;
(6) buffer B: 0.1mol/L Tris-HCl and the mixing of 0.002 mol/L EDTA equal-volume.
6. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in step c, The 12 passage liquid-transfering guns using range 1000ul add buffer A.
7. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in step c, Use thermal cycler 95 DEG C heating 10min.
8. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in Step d, Use and on earthquake device, shake 5min, make buffer A and buffer B be sufficiently mixed.
9. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that in step e, Reaction orifice plate uses 96 hole PCR plate, and liquid pipettes the 12 passage liquid-transfering guns preferably employing range 1000ul.
10. high flux as claimed in claim 2 extracts Semen Maydis or the method for radish gene group DNA, it is characterised in that step e In, PCR reaction system adds 0.1% bovine serum albumin and 1% polyvinylpyrrolidone.
CN201610672468.4A 2016-08-16 2016-08-16 Method for extracting corn or radish genome DNA with high flux Withdrawn CN106011132A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610672468.4A CN106011132A (en) 2016-08-16 2016-08-16 Method for extracting corn or radish genome DNA with high flux

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610672468.4A CN106011132A (en) 2016-08-16 2016-08-16 Method for extracting corn or radish genome DNA with high flux

Publications (1)

Publication Number Publication Date
CN106011132A true CN106011132A (en) 2016-10-12

Family

ID=57128035

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610672468.4A Withdrawn CN106011132A (en) 2016-08-16 2016-08-16 Method for extracting corn or radish genome DNA with high flux

Country Status (1)

Country Link
CN (1) CN106011132A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107201363A (en) * 2017-07-24 2017-09-26 江苏大学 A kind of simplicity of DNA of plants, rapid extracting method
CN110184266A (en) * 2019-06-03 2019-08-30 中国农业科学院植物保护研究所 Citrus leaf DNA rapid extracting method and its application in Citrus Huanglongbing pathogen detection

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1308130A (en) * 2000-12-26 2001-08-15 李永武 Method of extracting nucleic and from microbe cell
CN103436524A (en) * 2013-06-17 2013-12-11 华南农业大学 Method for batch extraction of rice endosperm DNA

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1308130A (en) * 2000-12-26 2001-08-15 李永武 Method of extracting nucleic and from microbe cell
CN103436524A (en) * 2013-06-17 2013-12-11 华南农业大学 Method for batch extraction of rice endosperm DNA

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
陈平华等: "碱裂解叶片两步快速制备PCR模板技术研究", 《热带作物学报》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107201363A (en) * 2017-07-24 2017-09-26 江苏大学 A kind of simplicity of DNA of plants, rapid extracting method
CN110184266A (en) * 2019-06-03 2019-08-30 中国农业科学院植物保护研究所 Citrus leaf DNA rapid extracting method and its application in Citrus Huanglongbing pathogen detection
CN110184266B (en) * 2019-06-03 2021-06-04 中国农业科学院植物保护研究所 Rapid extraction method of citrus leaf DNA and application of rapid extraction method in citrus yellow shoot detection

Similar Documents

Publication Publication Date Title
Taylor et al. A high-throughput platform for the production and analysis of transgenic cassava (Manihot esculenta) plants
CA2888143C (en) Embryo sampling for molecular analysis
CN107385071A (en) Molecular labeling primer and application for Kiwi berry mill mountain system row Male cultivar identification
Wang et al. Molecular cell biology of male meiotic chromosomes and isolation of male meiocytes in Arabidopsis thaliana
CN106609298B (en) The reagent set and the climing long molecule of giant pumpkin of identification or the auxiliary identification climing long character of giant pumpkin mark
Zheng et al. Non-destructive high-throughput DNA extraction and genotyping methods for cotton seeds and seedlings
CN113430213A (en) Gene and method for regulating and controlling tomato lateral branches
CN101586102A (en) Genomic DNA extraction method of peanut leaf blades
CN106011132A (en) Method for extracting corn or radish genome DNA with high flux
CN107201363A (en) A kind of simplicity of DNA of plants, rapid extracting method
CN101967521B (en) PCR identification method for upland cotton HB red flower germplasm material
CN106755572A (en) I types DHV and duck plague virus double fluorescent quantitative PCR method
CN103276054A (en) Primer for auxiliary detection of soybean hundred-grain weight, and detection method thereof
CN106661574A (en) Methods and devices involving oil matrices
CN106604636B (en) Plant embryo storage and manipulation
CN104404161B (en) The multiplex PCR examination detection primer of transgenic corns and detection method
CN105087550A (en) Method for rapid and high-flux extraction of plant genome DNA and application of plant genome DNA
CN104357576B (en) A kind of Specific primer pair identifying rice varieties fine horse excellent 522 and parent thereof
CN103421776A (en) Genome-specific molecular marker primer of annual diploid dasypyrum villosum and application of primer
CN113462807B (en) SNP molecular marker and application thereof in improving barley salt tolerance
JP2019187256A (en) Channel chip for plant substance detection
Ludwig et al. Rice gene knockout or downregulation through CRISPR-cas9
CN117551742A (en) Melon DNA extraction method and identification method suitable for KASP detection
CN104164496A (en) Method for plant PCR detection based on preservation of DNA in FTA card
CN117981672A (en) Agricultural molecule breeding accurate auxiliary system

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20161012

WW01 Invention patent application withdrawn after publication